US9453241B2 - AAV-mediated subcellular targeting of heterologous rhodopsins in retinal ganglion cells - Google Patents

AAV-mediated subcellular targeting of heterologous rhodopsins in retinal ganglion cells Download PDF

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US9453241B2
US9453241B2 US13/696,252 US201113696252A US9453241B2 US 9453241 B2 US9453241 B2 US 9453241B2 US 201113696252 A US201113696252 A US 201113696252A US 9453241 B2 US9453241 B2 US 9453241B2
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Zhuo-Hua Pan
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Definitions

  • the present invention in the field of molecular biology and medicine relates to the targeting of microbial-type rhodopsins, such as the light-gated cation-selective membrane channel, channelrhodopsin-2 (Chop2 or ChR2) or the ion pump halorhodopsin (HaloR) in retinal ganglion cells as a basis for restoring visual perception and various aspects of vision.
  • microbial-type rhodopsins such as the light-gated cation-selective membrane channel, channelrhodopsin-2 (Chop2 or ChR2) or the ion pump halorhodopsin (HaloR) in retinal ganglion cells as a basis for restoring visual perception and various aspects of vision.
  • Vision normally begins when rods and cones (photoreceptors) convert light signals to electrical signals that are then relayed through second- and third-order retinal neurons and the optic nerve to the lateral geniculate nucleus and, then to the visual cortex where visual images are formed (Baylor, D, 1996, Proc. Natl. Acad. Sci. USA 93:560-565; Wässle, H, 2004, Nat. Rev. Neurosci. 5:747-57).
  • the severe loss of photoreceptor cells can be caused by congenital retinal degenerative diseases, such as retinitis pigmentosa (RP) (Sung, C H et al., 1991 , Proc. Natl. Acad. Sci.
  • RP retinitis pigmentosa
  • Age-related macular degeneration also results from degeneration and death of photoreceptor cells, which can cause severe visual impairment within the centrally located best visual area of the visual field.
  • the present inventor's work utilizes microbial-type rhodopsins that are similar to bacteriorhodopsin (Oesterhelt, D et al., 1973 , Proc. Natl. Acad. Sci. USA 70:2853-7), whose conformation change is caused by reversible photoisomerization of their chromophore group, all-trans retinaldehyde, and is directly coupled to ion movement through the membrane (Oesterhelt, D., 1998 , Curr. Opin. Struct. Biol. 8:489-500).
  • Chlamydomonas reinhardtii Nagel, G. et al., 2002 , Science 296:2395-8; Sineshchekov, O A et al., 2002 , Proc. Natl. Acad. Sci. USA 99:8689-94; Nagel, G. et al., 2003 , Proc. Natl. Acad. Sci. USA 100, 13940-45
  • Chop1 and Chop2 Two microbial-type opsins, channelopsin-1 and -2 (Chop1 and Chop2), have been cloned from Chlamydomonas reinhardtii (Nagel, G. et al., 2002 , Science 296:2395-8; Sineshchekov, O A et al., 2002 , Proc. Natl. Acad. Sci. USA 99:8689-94; Nagel, G. et al., 2003 , Proc. Natl. Acad. Sci. USA 100, 13940
  • Chop2 a seven transmembrane domain protein, becomes photo-switchable when bound to the chromophore all-trans retinal. Chop2 is particularly attractive because its functional light-sensitive channel, channelrhodopsin-2 (Chop2 retinalidene abbreviated ChR2) with the attached chromophore is permeable to physiological cations. Unlike animal rhodopsins, which only bind the 11-cis conformation, Chop2/ChR2 binds all-trans retinal isomers, obviating the need for all-trans to 13-cis isomerization supplied by the vertebrate visual cycle.
  • ChR2 Chop2 retinalidene abbreviated ChR2
  • ChR2 (Lagali et al., Nat. Neurosci. 11:667-675 (2008); NpHR by (Busskamp V. et al., Science 329, 413-417 (2010); synthetically engineered potassium (SPARK) and glutamate (LiGIuR) channels (Greenberg, K P et al., Invest. Ophthalmol. Vis. Sci. 47, 4750 (2006; abstract); Kolstad et al., Invest. Ophthalmol. Vis. Sci 49:3897 (2009; Abstract) and the G protein-coupled receptor melanopsin (Lin, B. et al., Proc. Natl. Acad. Sci. USA 105:16009-16014 (2008)) in normally nonphotosensitive bipolar, amacrine, and ganglion cells or nonfunctional photoreceptors.
  • AAV2 adeno-associated virus
  • HaloR-expressing RGCs respond to light with rapid hypopolarization and suppression of spike activity. After termination of the light stimulus, their membrane potential exhibited a rapid rebound overshoot with robust sustained or transient spike firing.
  • Coexpression of ChR2/HaloR in RGCs produced ON, OFF, and even ON-OFF responses, depending on the wavelength of the light stimulus. Suggesting that the expression of multiple microbial rhodopsins such as ChR2 and HaloR is a possible strategy to restore both ON and OFF light responses in the retina after the death of rod and cone photoreceptors.
  • the present invention is a refinement and significant step forward of the inventor's prior work, being directed to differential, subcellular “site-selective expression” of these light-sensor-encoding nucleic acids by adding sorting or targeting motifs to the vectors that confer such selectivity. This adds to the “spatial resolution” of vision restoration achieved in this manner in those suffering vision loss or blindness caused, for example, by any of a number of retinal degenerative diseases.
  • the present inventor's approach does not require, introducing exogenous cells and tissues or physical devices, thus avoiding obstacles encountered by existing approaches, though the combined use of the present approach with visual prostheses or devices is also envisioned.
  • the present inventor has discovered that differentially targeted expression of ChR2 and HaloR to different subcellular regions in RGCs recreates the antagonistic center-surround receptive field in these cells that further permits improvement of the visual spatial processing for restored vision.
  • the primary spatial distinction of expression is in center vs. peripheral regions of the cells. Peripheral is also referred to in the art as the “surround” or as “off center,” terms that are well understood.
  • RGCs are rendered light sensitive by expression of ChR2 and/or HaloR selectively in somatodendritic region while being kept to a minimum in the axonal region. This enables maintenance of visual spatial processing.
  • sorting motifs also referred to here as “targeting motifs, “sorting sequences” or “targeting sequences” present in a vector that comprises the light sensor encoding nucleic acid.
  • Such a motif mediates site- or region-selective expression of the ChR2 or HaloR in subcellular regions of a retinal neuron, preferably an RGC.
  • This targeting serves as a basis for enhanced spatial control and specificity, and results in transmission of appropriate signals, providing better contrast, which more closely resembling signals from a healthy, intact retina, to higher centers of the visual cortex to compensate for damage and degeneration in retinal photoreceptors.
  • the present invention is directed to a nucleic acid molecule encoding a rhodopsin for differential expression in subcellular regions of a retinal neuron, preferably an RGC, which molecule comprises:
  • the nucleic promoter and regulator sequence comprise a cytomegalovirus enhancer/chicken ⁇ -actin promoter (CAG), preferably SEQ ID NO:26, and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), preferably SEQ ID NO:27, and (d) is preferably SEQ ID NO:28.
  • CAG cytomegalovirus enhancer/chicken ⁇ -actin promoter
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • the nucleic acid molecule may further comprise, linked in frame with (a) and (b), a third nucleotide sequence encoding a reporter polypeptide, preferably GFP; a preferred sequence is SEQ ID NO:25.
  • the light-gated channel rhodopsin is preferably ChR2, such as SEQ ID:22, or a biologically active fragment, most preferably SEQ ID NO: 22.
  • the light driven ion pump rhodopsin is preferably HaloR, most preferably SEQ ID NO:24.
  • the sorting motif is one that targets the center of the neuron's receptive field, for example, to one or more of the following subcellular regions: the soma, the proximal dendritic region, or the axon initial segment.
  • Preferred sorting motif-encoding sequences are a nucleotide sequence encoding (a) voltage-gated potassium channel 2.1 (Kv2.1), which is or comprises SEQ ID NO:1; or (b) the ankyrin binding domain of voltage-gated sodium channel 1.6 (Nav1.6), which is or comprises SEQ ID NO:3.
  • the encoded amino acid sequence of the motif is preferably (a) the sequence of Kv2.1, which is or comprises SEQ ID NO:2; or (b) the sequence of the ankyrin-binding domain of Nav1.6, which is or comprises SEQ ID NO:4.
  • the motif is one that targets the rhodopsin ( ⁇ the reporter gene) to the surround or off-center part of the neuron's receptive field, for example, to the somatodendritic region of the neurons.
  • Preferred sorting motif-encoding sequences are a nucleotide sequence encoding (a) the cytoplasmic C-terminal segment of neuroligin-1 (NLG-1), which is or comprises SEQ ID NO:5; or (b) the myosin binding domain of melanophilin (MLPH), which is or comprises SEQ ID NO:7.
  • the encoded amino acid sequence of the motif is preferably (a) the sequence of the cytoplasmic C-terminal segment of NLG-1 which is or comprises, SEQ ID NO:6; or (b) the sequence of the myosin-binding domain of MLPH, which is or comprises SEQ ID NO:8.
  • a recombinant adeno-associated virus expression vector preferably an AAV2 vector, comprising any of the above nucleic acid molecules.
  • the sequence of the nucleic acid molecule is flanked at its 5′ end by a 5′ inverted terminal repeat (ITR) and at its 3′ end by a 3′ ITR of the AAV, preferably AAV2.
  • ITR inverted terminal repeat
  • the sequence of these ITR is preferably SEQ ID NO:17 and SEQ ID NO:18, respectively.
  • the sorting motif is one that targets the center of the neuron's receptive field.
  • a preferred nucleotide sequence encoding the motif is (a) the sequence encoding Kv2.1, which is or comprises SEQ ID NO:1; or (b) the sequence encoding the ankyrin binding domain of Nav1.6, which is or comprises SEQ ID NO:3.
  • the amino acid sequence of the encoded motif is (a) the acid sequence of Kv2.1, which is or comprises SEQ ID NO:3; or (b) the sequence of the ankyrin binding domain of Nav1.6, which is or comprises SEQ ID NO:4.
  • the sorting motif is one that targets the surround or off-center of the neuron's receptive field.
  • the motif is selected from the group consisting of nucleotide sequence encoding (a) the cytoplasmic C-terminal segment of NLG-1, which is or comprises SEQ ID NO:5; or (b) myosin binding domain of MLPH, which is or comprises SEQ ID NO:7.
  • the amino acid sequence of the encoded motif is (a) the sequence of the cytoplasmic C-terminal segment NLG-1, which is or comprises SEQ ID NO:6; or (b) the sequence of the myosin-binding domain of MLPH, which is or comprises SEQ ID NO:8.
  • the above expression vector can have one of the following schematic structures:
  • a preferred expression vector for targeting ChR2 to the center of the neuron's receptive field has the schematic structure and nucleotide sequence selected from the following group
  • SEQ ID NO: 32 (a) 5′- ITR -CAG-ChR2-GFP- ⁇ Kv2.1 Motif)-WPRE- bGHpolyA- ITR -3′,; SEQ ID NO: 33 (b) 5′- ITR -CAG-ChR2- ⁇ Kv2.1 Motif)-WPRE-bGHpolyA- ITR -3′,; SEQ ID NO: 34 (c) 5′- ITR -CAG-ChR2-GFP- ⁇ Nav2.6 Motif)-WPRE- bGHpolyA- ITR -3′, and; SEQ ID NO: 35 (d) 5′- ITR -CAG-ChR2- ⁇ Nav2.6 Motif)-WPRE-bGHpolyA- ITR -3′,.
  • a preferred expression vector for targeting ChR2 to the surround or off-center of the neuron's receptive field has the schematic structure and nucleotide sequence selected from the following group
  • SEQ ID NO: 36 (a) 5′- ITR -CAG-ChR2-GFP- ⁇ NLG-1 Motif)-WPRE- bGHpolyA- ITR -3′,; SEQ ID NO: 37 (b) 5′- ITR -CAG-ChR2- ⁇ NLG-1 Motif)-WPRE-bGHpolyA- ITR -3′,; SEQ ID NO: 38 (c) 5′- ITR -CAG-ChR2-GFP- ⁇ MLPH Motif)-WPRE- bGHpolyA- ITR -3′, and; SEQ ID NO: 39 (d) 5′- ITR -CAG-ChR2- ⁇ MLPH Motif)-WPRE-bGHpolyA- ITR -3′,.
  • a preferred expression vector targeting HaloR to the center of the neuron's receptive field has the schematic structure and nucleotide sequence selected from the following group:
  • SEQ ID NO: 40 (a) 5′- ITR -CAG-HaloR-GFP- ⁇ Kv2.1 Motif)-WPRE- bGHpolyA- ITR -3′,; SEQ ID NO: 41 (b) 5′- ITR -CAG-HaloR- ⁇ Kv2.1 Motif)-WPRE-bGHpolyA- ITR -3′,; SEQ ID NO: 42 (c) 5′- ITR -CAG-HaloR- ⁇ Nav2.6 Motif)-WPRE- bGHpolyA- ITR -3′, and; SEQ ID NO: 43 (d) 5′- ITR -CAG-HaloR-GFP- ⁇ Nav2.6 Motif)-WPRE- bGHpolyA- ITR -3′,;
  • a preferred expression vector for targeting HaloR to the surround or off-center of the neuron's receptive field has the schematic structure and nucleotide sequence selected from the following group
  • SEQ ID NO: 44 (a) 5′- ITR -CAG-HaloR-GFP- ⁇ NLG-1 Motif)-WPRE- bGHpolyA- ITR -3′,; SEQ ID NO: 45 (b) 5′- ITR -CAG-HaloR- ⁇ NLG-1 Motif)-WPRE-bGHpolyA- ITR -3′,; SEQ ID NO: 46 (c) 5′- ITR -CAG-HaloR-GFP- ⁇ MLPH Motif)-WPRE- bGHpolyA- ITR -3′, and; SEQ ID NO: 47 (c) 5′- ITR -CAG-HaloR- ⁇ MLPH Motif)-WPRE-bGHpolyA- ITR -3′,.
  • the above expression vector further comprises AAV vector backbone nucleotide sequence SEQ ID NO:29 linked to the 3′ end of the AAV 3′ITR sequence.
  • the present invention is directed to a method of restoring light sensitivity to a retina, comprising:
  • the desired subcellular site is soma, proximal dendritic region, or axon initial segment, where preferably the motif is one that targets the rhodopsin to the center of the RGCs receptive field.
  • the desired subcellular site is the somatodendritic region, where preferably the motif is one that targets the surround or off-center of the RGCs receptive field.
  • the nucleic acid molecule comprises any of the molecules above and the vector is the any of expression vectors above.
  • FIG. 1 is a group of photomicrographs comparing fluorescence intensity (originally green, converted to white, on black background) from green fluorescent protein (GFP) encoded in frame with ChR2 with or without (control) a sorting motif.
  • the sorting motifs tested as indicated in abbreviated form in the panels (described in more detail elsewhere in this document), were: Kv2.1, Nav1.6, AMPAR, Kv4.2, MLPH, nAchR, NGL-1 AND TLCN.
  • the arrow-heads in each panel point to the axon of the ChR2-GFP expressing RGCs. The results appear in tabular form in Table 2, below.
  • the present inventors discovered that certain protein sorting motifs used in AAV-mediated transduction direct targeted expression of Chop2 or HaloR or, for visualization, a test reporter gene (Green fluorescent protein, GFP) to RGCs results in differential expression of the targeted reporter gene in different compartments or subcellular sites of the RGCs.
  • a test reporter gene Green fluorescent protein, GFP
  • the present Examples show differential expression of ubiquitously expressing light sensitive channels, namely ChR2 driven by the CAG promoter and under the influence of various targeting motifs in distinct subcellular regions or sites of retinal ganglion cells.
  • a depolarizing light sensor such as ChR2
  • ON type retinal neurons ON type ganglion cells and/or ON type bipolar cells
  • a hypopolarizing light sensor such as HaloR (a chloride pump)
  • HaloR a chloride pump
  • the followings approaches used to restore the light sensitivity of inner retinal neurons are enhanced by the use, disclosed herein, of peptide/polypeptide sorting motifs expressed using recombinant vectors in selected subcellular sites/regions of retinal neurons, particularly RGC.
  • Ubiquitously expressing light sensitive channels such as ChR2 are employed to produced membrane depolarization in all types of ganglion cells (both ON and OFF ganglion cells), or all types of bipolar cells (rod bipolar cells, and ON and OFF cone bipolar cells).
  • the AAV vector with CAG promoter has already partially achieved this approach in rodent retinas, as exemplified herein.
  • a depolarizing light sensor such as ChR2
  • ON type retinal neurons such as ON type ganglion cells or ON type bipolar cells.
  • Fragments of a human gap junctional protein (connexin-36) promoter were found to target GFP in ON-type retinal ganglion cells by using AAV-2 virus vector (Greenberg K P et al., 2007 , ARVO abstract, 2007).
  • a readily packable shorter version of mGluR6 promoter of ( ⁇ 2.5 kb) would allow targeting of ChR2 to ON type bipolar cells (both rod bipolar cells and ON type cone bipolar cells).
  • Cell specific promoters are used to target the specific types of retinal neurons.
  • a promoter that could target rod bipolar cells is Pcp2 (L7) promoter (Tomomura, M et al., 2001 , Eur J Neurosci. 14:57-63).
  • the length of the active promoter is preferably less than 2.5 Kb so it can be packaged into the AAV viral cassette.
  • a depolarizing light sensor such as ChR2 is targeted to ON type ganglion cells or ON type cone bipolar cells and a hypopolarizing light sensor, such as halorhodopsin, to OFF type ganglion cells or OFF type cone bipolar cells to create ON and OFF pathways.
  • a hypopolarizing light sensor such as halorhodopsin
  • an adequately short (packable) version of mGluR6 promoter ⁇ 2.5 kb
  • the Neurokinin-3 (NK-3) promoter would be used to target halorhodopsin to OFF cone bipolar cells (Haverkamp, S et al., 2002 , J Compar. Neurol. 455:463-76.
  • a depolarizing light sensor such as ChR2 is targeted to rod bipolar cells and their target AII amacrine cells, an ON type retinal cell (which communicate with ON and OFF cone bipolar cells).
  • Table 1 describes the sorting peptide/polypeptide motifs examined by the present inventors presenting both the nucleotide and amino acid sequences, and a conclusion about their effects on sorting or targeting of the linked encoded proteins to different subcellular sites.
  • Sorting Motif (Receptive Field) Kv2.1 Voltage-gated potassium Cytoplasmic Proximal dendrites, soma channel 2.1 1 C-terminus (center) aa sequence: (SEQ ID NO: 2) nt sequence: (SEQ ID NO: 1) QSQPILNTKEMAPQSKPPEELEMSSM CAG TCT CAG CCC ATC CTG AAC ACT AAG GAG ATG GCC PSPVAPLPARTEGVIDMRSMSSIDSF CCT CAG AGT AAA CCC CCT GAG GAA CTG GAA ATG AGC ISCATDFPEATRF (65) TCC ATG CCA TCT CCA GTG GCT CCT CTG CCA GCT AGG ACC GAG GGC GTG ATT GAC ATG AGA GAC ATG TCT AGT ATC GAT AGC TTC ATT TCC TGC GCC ACC GAC TTC CCC GAA GCT
  • Motif the functional name or location of each motif.
  • Subcellular targeted site the reported site of preferential subcellular targeting.
  • Receptive Field the central vs. surround (off-center or peripheral) region of the cell Superscripted numbers refer to the following references: 1 Lim ST, et al. . Neuron. 25: 385-97 (2000). 2 Garrido, J. et al. Science 300: 2091 (2003). 3 Bioko, T. et al., J. Neurosci. 232306-2313 (2003). 4 Rosales, C. et al. Eur. J. Neurosci. 22, 2381-2386 (2005). 5 Lewis, T. et al. Nat. Neurosci. 12, 568-576 (2009).
  • a depolarizing light sensor such as ChR2
  • a hypopolarizing light sensor such as HaloR in selected subcellular sites of OFF type retinal neurons (OFF type RGC and/or OFF type bipolar cells)
  • OFF type RGC and/or OFF type bipolar cells could create even more useful ON and OFF pathways in photoreceptor degenerated retinas that is possible without the selective targeting mediated by the sorting motifs described here.
  • a preferred embodiment would be:
  • Such combined treatment would enhance not only signal transmission but contrast and hence visual resolution in such molecularly enhanced or modified cells. This more closely resembles the physiological effects of signals transmitted to these cells by retinal photoreceptors in a normal vision state.
  • Such specificity and selectivity would be aided by the use of ON cell-specific promoters and OFF cell-specific promoters compared to the ubiquitous promoters exemplified here. Once such promoters are identified, they would be inserted into the various vectors described here in place of CAG.
  • nucleic acid vectors may be used in these methods, e.g., recombinant viruses, such as recombinant adeno-associated virus (rAAV), recombinant adenoviruses, recombinant retroviruses, recombinant poxviruses, and other known viruses in the art, as well as plasmids, cosmids and phages, etc.
  • recombinant viruses such as recombinant adeno-associated virus (rAAV), recombinant adenoviruses, recombinant retroviruses, recombinant poxviruses, and other known viruses in the art, as well as plasmids, cosmids and phages, etc.
  • rAAV recombinant adeno-associated virus
  • retroviruses recombinant retroviruses
  • poxviruses recombinant poxviruses
  • Adeno-associated viruses are small, single-stranded DNA viruses which require a helper virus for efficient replication (Berns, K I, Parvoviridae: the viruses and their replication , p. 1007-1041 (vol. 2), in Fields, B N et al., Fundamental Virology, 3rd Ed., (Lippincott-Raven Publishers, Philadelphia (1995)).
  • the 4.7 kb genome of AAV has two inverted terminal repeats (ITR) and two open reading frames (ORFs) which encode the Rep proteins and Cap proteins, respectively.
  • the Rep reading frame encodes four proteins of molecular weights 78, 68, 52 and 40 kDa.
  • VP3 comprises >80% of total AAV virion proteins.
  • Flanking the rep and cap ORFs at the 5′ and 3′ ends are 145 bp ITRs, the first 125 bps of which can form Y- or T-shaped duplex structures.
  • the two ITRs are the only cis elements essential for AAV replication, rescue, packaging and integration of the genome.
  • Two conformations of AAV ITRs called “flip” and “flop” exist (Snyder, R O et al., 1993 , J Virol., 67:6096-6104; Berns, K I, 1990 Microbiol Rev, 54:316-29).
  • the entire rep and cap domains can be excised and replaced with a transgene such as a reporter or therapeutic transgene (Carter, B J, in Handbook of Parvoviruses , P. Tijsser, ed., CRC Press, pp. 155-68 (1990)).
  • AAVs have been found in many animal species, including primates, canine, fowl and human (Murphy, F A et al., The Classification and Nomenclature of Viruses: Sixth Rept of the Int'l Comm on Taxonomy of Viruses, Arch Virol , Springer-Verlag, 1995).
  • Six primate serotypes are known (AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6) (and more are known that infect other classes of mammals).
  • the AAV ITR sequences and other AAV sequences employed in generating the minigenes, vectors, and capsids, and other constructs used in the present invention may be obtained from a variety of sources.
  • the sequences may be provided by any of the above 6 AAV serotypes or other AAV serotypes or other densoviruses, including both presently known human AAV and yet to yet-to-be-identified serotypes.
  • AAVs known to infect other animal species may be the source of ITRs used in the present molecules and constructs.
  • Capsids from a variety of serotypes of AAV may be combined in various mixtures with the other vector components (e.g., WO01/83692 (Hildiger et al.; U.S. Pat. No. 7,056,502; US Pat Pub. 2003/0013189 (Wilson et al).
  • WO01/83692 Hildiger et al.
  • U.S. Pat. No. 7,056,502 US Pat Pub. 2003/0013189
  • rAAV virions wherein the rAAV2 ITRs described herein are combined with AAV5 capsid proteins.
  • Such constructs may be advantageous because humans are less likely to have been pre-exposed to AAV5 vs.
  • AAV2 and therefore are less likely to have immunological memory (e.g., circulating antibodies or capsid-specific T lymphocytes).
  • immunological memory e.g., circulating antibodies or capsid-specific T lymphocytes.
  • WO2005/021768 (Tak et al.); Adriaansen, J et al., Ann Rheum Dis 2005, 64:1677-1684; US Pat. Pub. 2004-072351 (Womer et al.); U.S Pat. Pub. 2005/0255089 (Chiorini et al.), Adriaansen, J et al., Ann Rheum Dis 2005, 64:1677-1684, all of these references concerning rAAV are incorporated by reference in their entirety.
  • the present invention includes AAV2 ITR's combined with capsid proteins of any of 6 known primate AAV serotypes. It is also known in the art that certain mutations in capsid proteins can enhance transfection efficiency, and it would within the ordinary skill of the art to test and select appropriate mutations for use in the present invention. Many of these viral strains or serotypes are available from the American Type Culture Collection (ATCC), Manassas, Va., or are available from a variety of other sources (academic or commercial).
  • ATCC American Type Culture Collection
  • Manassas, Va. or are available from a variety of other sources (academic or commercial).
  • sequences used in preparing the vectors and viruses of the invention may be synthesized using known techniques, based on published AAV sequences, e.g., available from a variety of databases.
  • the source of the sequences utilized to prepare the present constructs is not considered to be limiting.
  • the selection of the AAV serotype and species (of origin) is within the skill of the art and is not considered limiting.
  • the rAAV construct e.g., a minigene or cassette
  • the rAAV minigene is formed by AAV ITRs and a heterologous nucleic acid molecule for delivery to a host cell.
  • the minigene comprises ITRs, most preferably AAV2 ITRs, located 5′ and 3′ to the heterologous sequence (rhodopsin protein and targeting sequence) being expressed.
  • Vectors comprising 5′ ITR and 3′ ITR sequences arranged in tandem, e.g., 5′ to 3′ or a head-to-tail, or in another configuration may also be useful.
  • inventions include a minigene with multiple copies of the ITRs, or one in which 5′ ITRs (or conversely, 3′ ITRs) are located both 5′ and 3′ to the heterologous sequence.
  • the ITRs sequences may be located immediately upstream and/or downstream of the heterologous sequence; intervening sequences may be present.
  • the preferred ITRs are from AAV2, but they may also originate from AAV5 or from any other AAV serotype.
  • the present construct or minigene may include 5′ ITRs from one serotype and 3′ ITRs from another.
  • the AAV sequences used are preferably the 140145 by cis-acting 5′ and 3′ ITR sequences (e.g., Carter, B J, supra). Preferably, the entire ITR sequence is used, although minor modifications are permissible. The most ITR's used in the present examples are
  • An example of such a molecule employed in the present invention is a “cis-acting” plasmid containing the heterologous sequence, preferably the ChR2 (any of SEQ ID NO:30-39) or HaloR sequence (any of SEQ ID NO:40-47, with or without an in-frame GFP sequence, with an in-frame sorting motif, promoter/regulatory sequences, all flanked by the 5′ and 3′ AAV ITR sequences.
  • the heterologous sequence preferably the ChR2 (any of SEQ ID NO:30-39) or HaloR sequence (any of SEQ ID NO:40-47, with or without an in-frame GFP sequence, with an in-frame sorting motif, promoter/regulatory sequences, all flanked by the 5′ and 3′ AAV ITR sequences.
  • the heterologous sequence encodes a protein or polypeptide which is desired to be delivered to and expressed in a cell and a targeting motif that differentially targets the polypeptide to particular subcellular regions of the cell, preferably an RGC.
  • the heterologous sequence is a nucleic acid molecule that functions as a transgene.
  • transgene refers to a nucleic acid sequence heterologous to the AAV sequence, and encoding a desired product, preferably ChR2 or HaloR plus the sorting motif, and the regulatory sequences which direct or modulate transcription and/or translation of this nucleic acid in a host cell, enabling expression in such cells of the encoded product.
  • Preferred polypeptide products are those that can be delivered to the eye, particularly to retinal neurons, most preferably to RGC.
  • the transgene/targeting sequence is delivered and differentially expressed in selected subcellular sites as directed by the sorting motif, in order to treat or otherwise improve the vision status of a subject with an ocular disorder.
  • the targeted ocular cells are preferably retinal neurons, namely, bipolar cells and most preferably, RGC.
  • the brightness of the light needed to stimulate evoked potential in transduced mouse retinas indicates that a channel opsin with increased light sensitivity may be more desirable. This can be achieved by selection of a suitable naturally occurring opsin, for example other microbial-type rhodopsins, or by modifying the light sensitivity of ChR2 as well as its other properties, such as ion selectivity and spectral sensitivity, to produce diversified light-sensitive channels to better fit the need for vision restoration.
  • transgenes may be used to encode separate subunits of a protein being delivered, or to encode different polypeptides the co-expression of which is desired. If a single transgene includes DNA encoding each of several subunits, the DNA encoding each subunit may be separated by an internal ribozyme entry site (IRES), which is preferred for short subunit-encoding DNA sequences (e.g., total DNA, including IRES is ⁇ 5 kB). Other methods which do not employ an IRES may be used for co-expression, e.g., the use of a second internal promoter, an alternative splice signal, a co- or post-translational proteolytic cleavage strategy, etc., all of which are known in the art.
  • IRES internal ribozyme entry site
  • the coding sequence or non-coding sequence of the present nucleic acids, including all domains to be expressed preferably are codon-optimized for the species in which they are to be expressed, particularly mammals and humans. Such codon-optimization is routine in the art.
  • transgene encodes a full length polypeptide, preferably ChR2
  • the present invention is also directed to vectors that encode a biologically active fragment of ChR2 (nucleotides: SEQ ID NO:19; amino acids: SEQ ID NO:20) or a (preferably conservative) amino acid substitution variant or mutant of ChR2, or a full length HaloR (nucleotide SEQ ID NO:23; amino acid SEQ ID NO:24) or a biologically active fragment, variant, mutant, or fusion/chimeric nucleic acid encoding a fusion protein.
  • CatCh calcium translocating channelrhodopsin (mutation at L132C) mediates an accelerated response time and a voltage response that is ⁇ 70-fold more light sensitive than that of wild-type ChR2; these properties stem from enhanced Ca2+ permeability.
  • a biologically active fragment or variant is a “functional equivalent”—a term that is well understood in the art and is further defined in detail herein.
  • the requisite biological activity of the encoded fragment or variant using any method disclosed herein or known in the art to establish activity of a channel opsin, has the following activity relative to the wild-type native polypeptide: about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, and any range derivable therein, such as, for example, from about 70% to about 80%, and more preferably from about 81% to about 90%; or even more preferably, from about 91% to about 99%.
  • amino acid sequence identity of the encoded polypeptide variants of the present invention are determined using standard methods, typically based on certain mathematical algorithms.
  • percent identity between two amino acid sequences is determined using the Needleman and Wunsch ( J. Mol. Biol. 48:444-453 (1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of Meyers and Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • nucleotide and amino acid sequences of the present invention can further be used as a “query sequence” to perform a search against public databases, for example, to identify other family members or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (Altschul et al. (1990) J. Mol. Biol. 215:403-10).
  • Gapped BLAST can be utilized (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402).
  • the default parameters of the respective programs e.g. XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See World Wide Web URL ncbi.nlm.nih.gov.
  • the preferred amino acid sequence variant has the following degrees of sequence identity with the native, full length channel opsin polypeptide, preferably Chop2 from C. reinhardtii (SEQ ID NO:_) or with a fragment thereof (e.g., SEQ ID NO:_): about 50%, about 55%, about 60%, about 65%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, and any range derivable therein, such as, for example, from about 70% to about 80%, and more preferably from about 81% to about 90%; or even more preferably, from about 91% to about 99% identity.
  • Any of a number of known recombinant methods are used to produce a DNA molecule encoding the fragment or variant.
  • Site-directed mutagenesis is a well-known technique for which protocols and reagents are commercially available (e.g., Zoller, M J et al., 1982 , Nucl Acids Res 10:6487-6500; Adelman, J P et al., 1983, DNA 2:183-93).
  • These mutations include simple deletions or insertions, systematic deletions, insertions or substitutions of clusters of bases or substitutions of single bases.
  • biologically functional equivalents it is well understood by those skilled in the art that, inherent in the definition of a “biologically functional equivalent” protein, polypeptide, gene or nucleic acid, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity.
  • Biologically functional equivalent peptides are thus defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted.
  • the shorter the length of the polypeptide the fewer amino acids changes should be made. Longer fragments may have an intermediate number of changes. The full length polypeptide protein will have the most tolerance for a larger number of changes. It is also well understood that where certain residues are shown to be particularly important to the biological or structural properties of a polypeptide residues in a binding regions or an active site, such residues may not generally be exchanged. In this manner, functional equivalents are defined herein as those poly peptides which maintain a substantial amount of their native biological activity.
  • hydropathy index of amino acids may also be considered in selecting variants.
  • Each amino acid has been assigned a hydropathy index on the basis of their hydrophobicity and charge characteristics, these are: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Glycine ( ⁇ 0.4); Thr ( ⁇ 0.7); Ser ( ⁇ 0.8); Trp ( ⁇ 0.9); Tyr ( ⁇ 1.3); Pro ( ⁇ 1.6); His ( ⁇ 12); Glu ( ⁇ 3.5); Gln ( ⁇ 3.5); Asp ( ⁇ 3.5); Asn ( ⁇ 3.5); Lys ( ⁇ 3.9); and Arg ( ⁇ 4.5).
  • hydropathy index in conferring interactive biological function on a proteinaceous molecule is generally understood in the art (Kyte and Doolittle, 1982 , J. Mol. Biol. 157:105-32). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score and still retain a similar biological activity. In making changes based upon the hydropathy index, the substitution of amino acids whose hydropathy indices are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred. It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biological functional equivalent polypeptide thereby created is intended for use in certain of the present embodiments.
  • U.S. Pat. No. 4,554,101 discloses that the greatest local average hydrophilicity of a proteinaceous molecule, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the molecule. See U.S. Pat. No. 4,554,101 for a hydrophilicity values.
  • the substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those which are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • the expression vector of the present invention includes appropriate sequences operably linked to the coding sequence(s) or ORF(s) to promote its expression in a targeted host cell.
  • “Operably linked” sequences include both expression control sequences such as. promoters that are contiguous with the coding sequences and expression control sequences that act in trans or distally to control the expression of the polypeptide product.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance nucleic acid or protein stability; and when desired, sequences that enhance protein processing and/or secretion.
  • RNA processing signals such as splicing and polyadenylation signals
  • sequences that stabilize cytoplasmic mRNA sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance nucleic acid or protein stability; and when desired, sequences that enhance protein processing and/or secretion.
  • Many varied expression control sequences including native and non-native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized herein, depending upon the type of expression desired.
  • Expression control sequences for eukaryotic cells typically include a promoter, an enhancer, such as one derived from an immunoglobulin gene, SV40, CMV, etc., and a polyadenylation sequence which may include splice donor and acceptor sites.
  • the polyadenylation (polyA) sequence generally is inserted 3′ to the coding sequence and 5′ to the 3′ ITR sequence.
  • the polyA from bovine growth hormone (bGH) is a suitable sequence and is abbreviated “bGHpolyA” (SEQ ID NO:28).
  • the regulatory sequences useful herein may also contain an intron, such as one located between the promoter/enhancer sequence and the coding sequence.
  • an intron such as one located between the promoter/enhancer sequence and the coding sequence.
  • One useful intron sequence is derived from SV40, and is referred to as the SV40 T intron sequence.
  • An IRES sequence may be used to produce more than one polypeptide from a single transcript.
  • An exemplary IRES is the poliovirus IRES which supports transgene expression in photoreceptors, RPE and ganglion cells.
  • the IRES is located 3′ to the coding sequence in the present vector, preferably an rAAV vector.
  • the promoter may be selected from a number of constitutive or inducible promoters that can drive expression of the selected transgene in an ocular setting, preferably in retinal neurons.
  • a preferred promoter is “cell-specific”, meaning that it is selected to direct expression of the selected transgene in a particular ocular cell type, such as photoreceptor cells.
  • a preferred constitutive promoters include the exemplified hybrid cytomegalovirus (CMV) immediate early enhancer/chicken ⁇ -actin promoter-exon 1-intron 1 element (together abbreviated as “CAG” SEQ ID NO:26, herein) used along with woodchuck hepatitis virus posttranscriptional regulatory element (abbreviated herein as “WPRE”; SEQ ID NO:27 herein).
  • CMV hybrid cytomegalovirus
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • RSV LTR promoter/enhancer examples include RSV LTR promoter/enhancer, the SV40 promoter, the CMV promoter, the dihydrofolate reductase (DHFR) promoter, and the phosphoglycerol kinase (PGK) promoter. Additional useful promoters are disclosed in W. W. Hauswirth et al., 1998, WO98/48027 and A. M. Timmers et al., 2000, WO00/15822.
  • Promoters that were found to drive RPE cell-specific gene expression in vivo include (1) a 528-bp promoter region (bases 1-528 of a murine 11-cis retinol dehydrogenase (RDH) gene (Driessen, C A et al., 1995 , Invest. Ophthalmol. Vis. Sci. 36:1988-96; Simon, A. et al., 1995 , J. Biol. Chem 270:1107-12, 1995; Simon, A.
  • RDH retinol dehydrogenase
  • Genomics 36:424-3 Genbank Accession Number X97752
  • a 2274-bp promoter region from a human cellular retinaldehyde-binding protein (CRALBP) gene
  • CRALBP retinaldehyde-binding protein
  • a 1485-bp promoter region from human RPE65 (Nicoletti, A et al., 1998 , Invest. Ophthalmol. Vis. Sci. 39, 637-44, Genbank Accession Number U20510).
  • promoters in WO00/15822 promoted RPE-cell-specific expression of GFP. It is envisioned that minor sequence variations in the various promoters and promoter regions discussed herein—whether additions, deletions or mutations, whether naturally occurring or introduced in vitro, will not affect their ability to drive expression in the cellular targets of the coding sequences of the present invention. Furthermore, the use of other promoters, even if not yet discovered, that are characterized by abundant and/or specific expression in retinal cells, particularly in bipolar or ganglion cells, is specifically included within the scope of this invention.
  • Another useful promoter is from a mGluR6 promoter-region of the Grm6 gene (GenBank accession number BC041684), a gene that controls expression of metabotropic glutamate receptor 6 ((Ueda Y et al., 1997 , J Neurosc. 17:3014-23).
  • the genomic sequence is shown in GenBank accession number—AL627215.
  • a preferred example of this promoter region sequence from the above GenBank record consists of 11023 nucleotides.
  • the original Umeda et al., study employed a 10 kb promoter, but the actual length of the promoter and the sequence that comprises control elements of Grm6 can be adjusted by increasing or decreasing the fragment length.
  • Another promoter is the Pcp2 (L7) promoter (Tomomura, M et al., 2001 , Eur J Neurosci. 14:57-63). Again, the length of the active promoter is preferably less than 2.5 Kb so it can be packaged into the rAAV viral cassette.
  • the neurokinin-3 (NK-3) promoter could be used to target HalorR to OFF cells (Haverkamp, S et al., 2002 , J Comparative Neurology, 455:463-76.
  • An inducible promoter is used to control the amount and timing of production of the transgene product in an ocular cell. Such promoters can be useful if the gene product has some undesired, e.g., toxic, effects in the cell if it accumulates excessively.
  • Inducible promoters include those known in the art, such as the Zn-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter; the T7 promoter; the ecdysone insect promoter; the tetracycline-repressible system; the tetracycline-inducible system; the RU486-inducible system; and the rapamycin-inducible system.
  • Any inducible promoter the action of which is tightly regulated and is specific for the particular target ocular cell type, may be used.
  • Other useful types of inducible promoters are ones regulated by a specific physiological state, e.g., temperature, acute phase, a cell's replicating or differentiation state.
  • One skilled in the art can select one or more expression control sequences, operably link them to the coding sequence being expressed to make a minigene, insert the minigene or vector into an AAV vector, preferably rAAV2, and cause packaging of the vector into infectious particles or virions following one of the known packaging methods for rAAV.
  • the rAAV2 used in the present invention may be constructed and produced using the materials and methods described herein and those well-known in the art.
  • the methods that are preferred for producing any construct of this invention are conventional and include genetic engineering, recombinant engineering, and synthetic techniques, such as those set forth in reference cited above.
  • AAV rep and cap sequences may be introduced into the host cell in any known manner including, without limitation, transfection, electroporation, liposome delivery, membrane fusion, biolistic deliver of DNA-coated pellets, viral infection and protoplast fusion.
  • Devices specifically adapted for delivering DNA to specific regions within and around the eye for the purpose of gene therapy have been described (for example, U.S. Pat. Pub. 2005/0277868, incorporated by reference) are used within the scope of this invention.
  • Such devices utilize electroporation and electromigration, providing, e.g., two electrodes on a flexible support that can be placed behind the retina.
  • a third electrode is part of a hollow support, which can also be used to inject the molecule to the desired area.
  • the electrodes can be positioned around the eye, including behind the retina or within the vitreous.
  • a useful nucleic acid molecule comprises, from 5′ to 3′, a promoter, an optional spacer between the promoter and the start site of the rep sequence, an AAV rep sequence, and an AAV cap sequence.
  • the rep and cap sequences are preferably provided in a single vector, though they may be provided separately in individual vectors.
  • the promoter may be any suitable constitutive, inducible or native promoter.
  • the delivery molecule that provides the Rep and Cap proteins may be in any form, preferably a plasmid which may contain other non-viral sequences, such as those to be employed as markers. This molecule typically excludes the AAV ITRs and packaging sequences. To avoid the occurrence of homologous recombination, other viral sequences, particularly adenoviral sequences, are avoided. This plasmid is preferably one that is stably expressed.
  • the rAAV may be produced using a triple transfection method with either the calcium phosphate (Clontech) or EffecteneTM reagent (Qiagen) according to manufacturer's instructions. See, also, Herzog et al., Nat. Med. 5:56-63 (1999).
  • the rAAV virions are produced by culturing host cells comprising a rAAV as described in Bi et al., supra, and WO2007/131180, which includes a rAAV construct to be packaged into a rAAV virion, an AAV rep sequence and an AAV cap sequence, all under control of regulatory sequences directing expression.
  • Suitable viral helper genes such as adenovirus E2A, E4Orf6 and VA, may be added to the culture preferably on separate plasmids. Thereafter, the rAAV virion which directs expression of the transgene is isolated in the absence of contaminating helper virus or wild type AAV.
  • a target cell may be infected in vitro, and the number of copies of the transgene in the cell monitored by Southern blots or quantitative PCR.
  • the level of RNA expression may be monitored by Northern blots quantitative RT-PCR.
  • the level of protein expression may be monitored by Western blot, immunohistochemistry, immunoassay including enzyme immunoassay (EIA) such as enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA) or by other methods.
  • EIA enzyme immunoassay
  • ELISA enzyme-linked immunosorbent assays
  • RIA radioimmunoassays
  • SEQ ID NO: 45 (same as above but without GFP) 5'-ITR—CAG—HaloR—(NLG-1 Motif)—WPRE—bGHpolyA—ITR-3' ttcatgcctt cttctttttc ctacagctcc tgggcaacgt gctggttatt gtgctgtctc ⁇ start HaloR atcattttgg caaagaatta agcttgagct cgcgatccgc agcc ATG ACT GAG ACA TTG CCA CCG GTA ACG GAA TCG GCT GTT GCG CTA CAG GCG GAG GTG ACC CAG AGG GAG CTG TTC GAG TTC GTT CTC AAC GAC CCC CTC CTC GCC AGT TCG CTG TAT ATT AAT ATC GCA CTG GCA GGG CTG TCG ATA CTG
  • the vectors that comprises the ChR2 or HaloR transgene and the targeting motifs disclosed herein for use to target retinal neurons as described above should be assessed for contamination using conventional methods and formulated into a sterile or aseptic pharmaceutical composition for administration by, for example, subretinal injection.
  • Such formulations comprise a pharmaceutically and/or physiologically acceptable vehicle, diluent, carrier or excipient, such as buffered saline or other buffers, e.g., HEPES, to maintain physiologic pH.
  • diluent such as buffered saline or other buffers, e.g., HEPES
  • HEPES buffered saline or other buffers
  • WO00/15822 For prolonged storage, the preparation may be frozen, for example, in glycerol.
  • the pharmaceutical composition described above is administered to a subject having a visual or blinding disease by any appropriate route, preferably by intravitreal or subretinal injection, depending on the retinal layer being targeted.
  • the DNA construct is targeted to either retinal ganglion cells or bipolar cells.
  • the ganglion cells are reasonably well-accessible to intravitreal injection.
  • Intravitreal and/or subretinal injection can provide the necessary access to the bipolar cells, especially in circumstances in which the photoreceptor cell layer is absent due to degeneration—which is the case in certain forms of degeneration that the present invention is intended to overcome.
  • a combination of a preferred promoter sequence linked to a reporter gene such as GFP or LacZ can be packaged into rAAV virus particles, concentrated, tested for contaminating adenovirus and titered for rAAV.
  • the right eyes of a number of test subjects, preferably inbred mice, are injected sub-retinally with about 1 ⁇ l of the rAAV preparation (e.g., greater than about 10 10 infectious units ml).
  • the right (test) and left (control) eyes of half the animals are removed, fixed and stained with an appropriate substrate or antibody or other substance to reveal the presence of the reporter gene.
  • a majority of the test retinas in injected eyes will exhibited a focal stained region, e.g., blue for LacZ/Xgal, or green for GFP consistent with a subretinal bleb of the injected virus creating a localized retinal detachment. All control eyes are negative for the reporter gene product. Reporter gene expression examined in mice sacrificed at later periods is detected for at least 10 weeks post-injection, which suggests persistent expression of the reporter transgene.
  • An effective amount of rAAV virions carrying a nucleic acid sequence according to this invention encoding the ChR2 or HaloR and targeting motif under the control of the promoter of choice, preferably CAG or a cell-specific promoter such as mGluR6, is preferably in the range of between about 10 10 to about 10 13 rAAV infectious units in a volume of between about 150 and about 800 ⁇ l per injection.
  • the rAAV infectious units can be measured according to McLaughlin, S K et al., 1988 , J Virol 62:1963. More preferably, the effective amount is between about 10 10 and about 10 12 rAAV infectious units and the injection volume is preferably between about 250 and about 500 ⁇ l.
  • dosages and volumes may be selected by the treating professional, taking into account the physical state of the subject (preferably a human), who is being treated, including, age, weight, general health, and the nature and severity of the particular ocular disorder.
  • boosters additional doses
  • a second treatment may be administered after 6 months or yearly, and may be similarly repeated.
  • Neutralizing antibodies to AAV are not expected to be generated in view of the routes and doses used, thereby permitting repeat treatment rounds.
  • the need for such additional doses can be monitored by the treating professional using, for example, well-known electrophysiological and other retinal and visual function tests and visual behavior tests.
  • the treating professional will be able to select the appropriate tests applying routine skill in the art. It may be desirable to inject larger volumes of the composition in either single or multiple doses to further improve the relevant outcome parameters.
  • the ocular disorders for which the present methods are intended and may be used to improve one or more parameters of vision include, but are not limited to, developmental abnormalities that affect both anterior and posterior segments of the eye.
  • Anterior segment disorders include glaucoma, cataracts, corneal dystrophy, keratoconus.
  • Posterior segment disorders include blinding disorders caused by photoreceptor malfunction and/or death caused by retinal dystrophies and degenerations.
  • Retinal disorders include congenital stationary night blindness, age-related macular degeneration, congenital cone dystrophies, and a large group of retinitis-pigmentosa (RP)-related disorders.
  • RP retinitis-pigmentosa
  • disorders include genetically pre-disposed dcath of photoreceptor cells, rods and cones in the retina, occurring at various ages.
  • severe retinopathies such as subtypes of RP itself that progresses with age and causes blindness in childhood and early adulthood
  • RP-associated diseases such as genetic subtypes of LCA, which frequently results in loss of vision during childhood, as early as the first year of life.
  • the latter disorders are generally characterized by severe reduction, and often complete loss of photoreceptor cells, rods and cones. (Trabulsi, E I, ed., Genetic Diseases of the Eye , Oxford University Press, NY, 1998).
  • this method is useful for the treatment and/or restoration of at least partial vision to subjects that have lost vision due to ocular disorders, such as RPE-associated retinopathies, which are characterized by a long-term preservation of ocular tissue structure despite loss of function and by the association between function loss and the defect or absence of a normal gene in the ocular cells of the subject.
  • ocular disorders such as RPE-associated retinopathies
  • a variety of such ocular disorders are known, such as childhood onset blinding diseases, retinitis pigmentosa, macular degeneration, and diabetic retinopathy, as well as ocular blinding diseases known in the art. It is anticipated that these other disorders, as well as blinding disorders of presently unknown causation which later are characterized by the same description as above, may also be successfully treated by this method.
  • the particular ocular disorder treated by this method may include the above-mentioned disorders and a number of diseases which have yet to be so characterized.
  • Visual information is processed through the retina through two pathways: an ON pathway which signals the light ON, and an OFF pathway which signals the light OFF (Wässle, supra). It is generally believed that the existence of the ON and OFF pathway is important for the enhancement of contrast sensitivity.
  • the visual signal in the ON pathway is relay from ON-cone bipolar cells to ON ganglion cells. Both ON-cone bipolar cells and ON-ganglion cells are depolarized in response to light.
  • the visual signal in the OFF pathway is carried from OFF-cone bipolar cells to OFF ganglion cells. Both OFF-cone bipolar cells and OFF-ganglion cells are hypopolarized in response to light.
  • Rod bipolar cells which are responsible for the ability to see in dim light (scotopic vision), are ON bipolar cells (depolarized in response to light). Rod bipolar cells relay the vision signal through AII amacrine cells (an ON type retinal cell) to ON an OFF cone bipolar cell.
  • AII amacrine cells an ON type retinal cell
  • Dissociated retinal cells and retinal slice are prepared, e.g., as described by Pan, Z.-H. J. Neurophysiol. 83 513-527 (2000); J. Cui, Y P et al., J. Physiol. 553:895-909 (2003). Recordings with patch electrodes in the whole-cell configuration can be made by an EPC-9 amplifier and PULSE software (Heka Electronik, Lambrecht, Germany).
  • Recordings are preferably made in Hanks' solution containing (in mM): NaCl, 138; NaHCO 3 , 1; Na 2 HPO 4 , 0.3; KCl, 5; KH 2 PO 4 , 0.3; CaCl 2 , 1.25; MgSO 4 , 0.5; MgCl 2 , 0.5; HEPES-NaOH, 5; glucose, 22.2; with phenol red, 0.001% v/v; adjusted to pH 7.2 with 0.3 N NaOH.
  • the electrode solution contains (in mM): K-gluconate, 133; KCl, 7; MgCl 2 , 4; EGTA, 0.1; HEPES, 10; Na-GTP, 0.5; and Na-ATP, 2; pH adjusted with KOH to 7.4.
  • the resistance of the electrode is about 13 to 15 M ⁇ . The recordings are performed at room temperature.
  • the multielectrode array recordings are on the procedures reported by Tian, N. et al., Neuron 39:85-96 (2003). Briefly, retinas are dissected and placed photoreceptor side down on a nitrocellulose filter paper strip. The mounted retina is placed in the MEA-60 multielectrode array recording chamber of 30 ⁇ m diameter electrodes spaced 200 ⁇ m apart (Multi Channel System MCS GmbH, Reutlingen, Germany), with the ganglion cell layer facing the recording electrodes. The retina is continuously perfused in oxygenated extracellular solution at 34° C.
  • the extracellular solution preferably contains (in mM): NaCl, 124; KCl, 2.5; CaCl 2 , 2; MgCl 2 , 2; NaH 2 PO 4 , 1.25; NaHCO 3 , 26; and glucose, 22 (pH 7.35 with 95% O 2 and 5% CO 2 ). Recordings are usually started 60 min after the retina is positioned in the recording chamber. The interval between onsets of each light stimulus is generally 10-15 s. The signals are filtered between 200 Hz (low cut off) and 20 kHz (high cut off). The responses from individual neurons are analyzed using, e.g., Offline Sorter software (Plexon, Inc., Dallas, Tex.).
  • Visual-evoked potential recordings are carried out, for example, in wild-type mice of the C57BL/6 and 129/Sy strains aged 4-6 months and in rd1/rd1 mice aged 6-11 months. Recordings are performed 2-6 months after viral vector injection. After general anesthesia, animals are mounted in a stereotaxic apparatus. Body temperature may be unregulated or maintained at 34° C. with a heating pad and a rectal probe. Pupils are dilated with 1% atropine and 2.5% accu-phenylephrine. A small portion of the skull ( ⁇ 1.5 ⁇ 1.5 mm) centered about 2.5 mm from the midline and 1 mm rostral to the lambdoid suture is drilled and removed.
  • Recordings are made from visual cortex (area V1) by a glass micropipette (resistance ⁇ 0.5 M after filling with 4 M NaCl) advanced 0.4 mm beneath the surface of the cortex at the contralateral side of the stimulated eye.
  • the stimuli are 20 ms pluses at 0.5 Hz.
  • Responses are amplified (1,000 to 10,000), band-pass filtered (0.3-100 Hz), digitized (1 kHz), and averaged over 30-250 trials.
  • light stimuli are generated by a 150 W xenon lamp-based scanning monochromator with bandwidth of 10 nm (TILL Photonics, Germany) and coupled to the microscope with an optical fiber.
  • light responses are evoked by the monochromator or a 175 W xenon lamp-based illuminator (Lambda LS, Sutter Instrument) with a band-pass filter of 400-580 nm and projected to the bottom of the recording chamber through a liquid light guider.
  • light stimuli are generated by the monochromator and projected to the eyes through the optical fiber.
  • the light intensity is attenuated by neutral density filters.
  • the light energy is measured by a thin-type sensor (TQ82017) and an optical power meter (e.g., Model: TQ8210, Advantest, Tokyo, Japan).
  • the present invention demonstrates a definite physiological recovery of function, which is expected to generate or improve various parameters of vision, including behavioral parameters.
  • Behavioral measures can be obtained using known animal models and tests, for example performance in a water maze, wherein a subject in whom vision has been preserved or restored to varying extents will swim toward light (Hayes, J M et al., 1993 , Behav Genet 23:395-403).
  • the transfection of retinal neurons with DNA encoding Chop2 provides residual retinal neurons, principally bipolar cells and ganglion cells, with photosensitive membrane channels.
  • a visual stimulus to the animal's visual cortex, the area of the brain responsible for processing visual signals; this therefore constitutes a form of vision, as intended herein.
  • vision may differ from forms of normal human vision and may be referred to as a sensation of light, also termed “light detection” or “light perception.”
  • vision is defined as the ability of an organism to usefully detect light as a stimulus for differentiation or action. Vision is intended to encompass:
  • the methods of the present invention that result in light perception, even without full normal vision, also improve or support normally regulated circadian rhythms which control many physiological processes including sleep-wake cycles and associated hormones. Although some blind individuals with residual RGCs can mediate their rhythms using RGC melanopsin, it is rare for them to do so. Thus, most blind persons have free-running circadian rhythms. Even when they do utilize the melanopsin pathway, the effect is very weak.
  • the methods of the present invention are thus expected to improve health status of blind individuals by enabling absent light entrainment or improving weakened (melanopsin-mediated) light entrainment of circadian rhythms which leads to better overall health and well-being.
  • the present invention provides a basis to improve deficits in other light-induced physiological phenomena.
  • Photoreceptor degeneration may result in varying degrees of negative masking, or suppression, of locomotor activity during the intervals in the circadian cycle in which the individual should be sleeping. Suppression of pineal melatonin may occur. Both contribute to the entrainment process.
  • improvement in these responses/activities in a subject in whom photoreceptors are or have degenerated contributes, independently of vision per se, to appropriate sleep/wake cycles that correspond with the subject's environment in the real world.
  • Yet another benefit of the present invention is normalization of pupillary light reflexes because regulation of pupil size helps modulate the effectivenees of light stimuli in a natural feed back loop.
  • the present invention promotes re-establishment of this natural feedback loop, making vision more effective in subject treated as described herein.
  • the present methods include the measurement of vision before, and preferably after, administering the present vector. Vision is measured using any of a number of methods well-known in the art or ones not yet established. Most preferred are:
  • the present invention enhances the vision of impaired subjects to such a level that by applying additional training methods, these individuals will achieve the above objectives.
  • Low sensitivity vision may emulate the condition of a person with a night blinding disorder, an example of which is Retinitis Pigmentosa (RP), who has difficulty adapting to light levels in his environment and who might use light amplification devices such as supplemental lighting and/or night vision devices.
  • RP Retinitis Pigmentosa
  • mice studied in the present Examples were rendered completely devoid of photoreceptors; this is quite rare, even in the worst human diseases.
  • the most similar human state is RP. In most cases of RP, central vision is retained till the very end. In contrast, in the studied mouse model, the mouse becomes completely blind shortly after birth.
  • Subjects with macular degeneration characterized by photoreceptor loss within the central “sweet spot” of vision (Macula Lutea), are expected to benefit by treatment in accordance with the present invention, in which case the resolution capability of the recovered vision would be expected to be higher due to the much higher neuronal density within the human macula.
  • the human vision system can operate over a 10 log unit range of luminance.
  • microbial type rhodopsins such as ChR2
  • the light conditions the patient encounters could fall outside of the operating range of the light sensor.
  • a light pre-amplification or attenuation device could be used to expand the operation range of the light conditions.
  • Such device would contain a camera, imaging processing system, and microdisplays, which can be assembled from currently available technologies, such as night vision goggles and/or 3D adventure and entertainment system. (See, for example the following URL on the Worldwide web—emagin.com/.)
  • the present invention may be used in combination with other forms of vision therapy known in the art. Chief among these is the use of visual prostheses, which include retinal implants, cortical implants, lateral geniculate nucleus implants, or optic nerve implants. Thus, in addition to genetic modification of surviving retinal neurons using the present methods, the subject being treated may be provided with a visual prosthesis before, at the same time as, or after the molecular method is employed.
  • the effectiveness of visual prosthetics can be improved with training of the individual, thus enhancing the potential impact of the ChR2 or HaloR transformation of patient cells as discussed herein.
  • An example of an approach to training is found in US 2004/0236389 (Fink et al.), incorporated by reference.
  • the training method may include providing a non-visual reference stimulus to a patient having a visual prosthesis based on a reference image.
  • the non-visual reference stimulus is intended to provide the patient with an expectation of the visual image that the prosthesis will induce.
  • Examples of non-visual reference stimuli are a pinboard, Braille text, or a verbal communication.
  • the visual prosthesis stimulates the patient's nerve cells, including those cells whose responsiveness has been improved by expressing ChR2 and/or HaloR as disclosed herein, with a series of stimulus patterns attempting to induce a visual perception that matches the patient's expected perception derived from the non-visual reference stimulus.
  • the patient provides feedback to indicate which of the series of stimulus patterns induces a perception that most closely resembles the expected perception.
  • the patient feedback is used as a “fitness function” (also referred to as a cost function or an energy function).
  • Subsequent stimuli provided to the patient through the visual prosthesis are based, at least in part, on the previous feedback of the patient as to which stimulus pattern(s) induce the perception that best matches the expected perception.
  • the subsequent stimulus patterns may also be based, at least in part, on a fitness function optimization algorithm, such as a simulated annealing algorithm or a genetic algorithm.
  • the method of improving or restoring vision in a subject further comprises training of that subject, as discussed above.
  • Preferred examples of training methods are:
  • an additional utility of restoring light sensitivity to inner retinal neurons in accordance with the present invention is the prevention or delay in the remodeling processes in the retina, and, possibly, in the higher centers.
  • Such retinal remodeling may have undesired consequences such as corruption of inner retinal network, primarily the connection between bipolar and RGCs.
  • the present methods would prevent or diminish the remodeling due to the lack of input; the present methods introduce this missing input (either starting from bipolar cells or ganglion cells), and thereby stabilize the retinal and higher visual center network.
  • the present invention would benefit other therapeutic approaches such as photoreceptor transplantation or device implants.
  • Adeno-associated virus serotype 2 (rAAV2) cassette carrying a channelopsin-2 and GFP (Chop2-GFP) fusion construct (Bi, A. et al. Neuron 50:23-33 (2006); WO2007/131180) were modified by inserting subcellular sorting motifs at the 3′ end of GFP (or, if no reporter is present, at the 3′ end of ChR2 or HaloR.
  • viral vectors carrying the transgene of ChR2-GFP-(motif) with a hybrid CMV early enhancer/chicken ( ⁇ -actin) promoter (CAG) were packaged and affinity purified at the Gene Transfer Vector Core of the University of Iowa.
  • mice 3-4 adult C57BL/6J mice aged 1-2 months per construct were used for the study.
  • the mice were anesthetized by intraperitoneal injection of ketamine (120 mg/kg) and xylazine (15 mg/kg). Under a dissecting microscope, a small perforation was made with a needle in the sclera region posterior to the limbus, and 1.0 ⁇ l of viral vector suspension at a concentration of >1 ⁇ 10 12 gv/ml was injected into the intravitreal space of each eye.
  • animals were sacrificed by CO2 asphyxiation followed by decapitation and enucleation.
  • Enucleated eyes were fixed in 4% paraformaldehyde in phosphate buffer (PB) for 20 minutes and the dissected retina flat mounted onto a microscope slide for histological studies.
  • the flat mounts were examined under a Zeiss Apotome microscope and Zstack images were taken at ⁇ 562 ms exposure time at optical sections of 1 ⁇ m apart in order to capture the axon, soma, and entire depth of the dendritic tree of each RGC.
  • Intensity profiles of axon, soma, and dendrites for each RGC were measured in ImageJ (obtained from NIH) by applying lines of width of 5 pixels.
  • ImageJ obtained from NIH
  • axon intensity profile was obtained by averaging 3 measurements
  • somatic intensity profile was obtained by averaging 3 measurements
  • dendritic intensity profile was obtained by averaging 9 measurements (3 proximal, 3 intermediate, and 3 distal).
  • Dendrite/axon (D/A) and soma/axon (S/A) intensity ratios were then calculated from the average values for each RGC.
  • Kv2.1 motif and targeted ChR2 Use of the Kv2.1 motif and targeted ChR2, and would similarly target HaloR, to soma and proximal dendritic regions (the center of receptive field) of RGCs.
  • Use of Nav1.6 motif targets to soma and axon initial segments (the center of the receptive field). Kv2.1 appears to achieve such targeting more effectively than does Nav1.6.
  • NLG-1 and MLPH sorting motifs targeted ChR2 (and would target HaloR) to distal dendritic regions (the surround of the receptive field) because, compared to control, they are more biased to distal dendritic regions. NLG appears to do this better.
  • Kv2.1, Nav1.6, NLG-1 and MLPH reduces expression of the ChR2 or HaloR in the axons of retinal ganglion cells.
  • the ankyrin binding domain of Nav1.6 preferentially targeted Chop2-GFP to the axon initial segments as well as decreased expression in the dendrites of RGCs with D/A ratio 4.5 fold less than control.
  • the overall fluorescence intensity was lower for Nav1.6 compared to the control which contributed to the lack of significant difference in the S/A ratio compared to control.
  • Motifs from nAchR, KV4.2, TLCN, and AMPAR did not show statistically significant differences from the control group in somatic fluorescence, D/A ratio, and S/A ratio in this study. However, it is believed that with varying conditions, further modified vectors, etc., these too are useful as sorting motifs for targeting of, and spatially selective expression of transduced ChR2 or HaloR in RGC.

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